Decellularizing Mammary Fat Pads: Deriving Extracellular Matrix Hydrogels from Mammary Fat Pads

First, place mammary fat pads or MFPs in a dish with Trypsin-EDTA solution and incubate for the desired duration to break the cell matrix adhesions. Wash the MFPs with deionized water and strain through a strainer. Place tissues in a beaker with a stir bar and add Triton X-100. Stir to lyse cells without affecting the growth factors in the tissue's extracellular matrix.

Strain the MFPs and rinse with deionized water. Transfer tissues back to the beaker, and add deoxycholic acid to lyse the remaining cells. Next, strain the MFPs and place in a beaker containing deionized water with penicillin-streptomycin to prevent contamination. Incubate the beaker at 4 degrees Celsius overnight.

Strain MFPs and place them into a beaker containing ethanol and peracetic acid. The peracetic acid increases the stiffness of the tissue's extracellular matrix. Stir for some time and repeat the straining step. Wash the MFPs with PBS and then with deionized water. Transfer the strained MFPs to a beaker containing propane-1-ol.

Stir for some time, strain the contents, and wash MFPs with deionized water. Dry the MFPs and store at minus 80 degrees Celsius. In the following protocol, we will perform the decellularization of murine mammary fat pads.

First, place the MFPs in six centimeter dishes with five milliliters of Trypsin-EDTA solution. Spray and wipe the dishes with 70% ethanol and incubate at 37 degrees Celsius for one hour. Use 0.7 millimeter strainers to wash the MFPs with deionized water by pouring water over the tissue three times. Use forceps to manually massage the tissue in between washes.

Next, briefly dry the tissue on a delicate task wipe and weigh it. Place the dried tissues in a pre-autoclaved beaker containing an appropriate sized stir bar, and cover the tissues with 60 milliliters of 3% t-octylphenoxy polyethoxyethanol per gram of tissue. Stir for one hour at room temperature. Then, dump the beakers contents into a strainer.

Rinse the beaker with deionized water and pour this onto the tissues. Repeat this rinsing process two more times, making sure to use forceps to manually massage the tissue in between rinses. After this, briefly dry the tissue on a delicate task wipe and weigh. Place the tissues and the stir bars back into the same beakers and cover them with 60 milliliters of 4% deoxycholic acid per gram of tissue. Stir at room temperature for one hour.

Next, dump the beaker's contents into a mesh strainer. Rinse the beaker with deionized water and pour this onto the tissues. Repeat this rinsing process two more times, making sure to use forceps to manually massage the tissue in between rinses. Briefly dry the rinsed tissue on a delicate task wipe and weigh it.

Place the dried tissues into the same beaker along with fresh deionized water supplemented with 1% penicillin streptomycin. Cover the beaker tightly with paraffin film and leave at 4 degrees Celsius overnight. The next day, drain the beaker contents into a strainer. Briefly dry the tissue on a delicate task wipe and weigh it. Then, place the MFPs in the same beaker with an appropriate sized stir bar.

Cover the tissue with 60 milliliters of a solution containing 4% ethanol and 0.1% peracetic acid per gram of tissue. Stir at room temperature for two hours. Dump the beaker's contents into a 0.7 millimeter strainer. Use forceps to manually massage the tissue and place the contents back into the beaker. Wash the tissue by covering it with 60 milliliters of 1X PBS per gram of tissue.

Stir at room temperature for 15 minutes. Repeat this entire washing process one more time. Next, dump the beaker's contents into a 0.7 millimeter strainer. Using forceps, manually massage the tissue and place the contents back into the beaker. Wash the tissue by covering it with 60 milliliters of deionized water per gram of tissue.

Stir at room temperature for 15 minutes. Repeat this entire washing process one more time. Briefly dry the washed tissue on a delicate task wipe and weigh it. Dump the tissue and contents into a strainer and use forceps to manually massage the tissue. Place the contents back into the beaker and cover the tissues with 60 milliliters of 100% n-propanol per gram of tissue.

Stir at room temperature for one hour. Then, briefly dry the tissue on a delicate task wipe and weigh it. Dump the tissue and contents into a 0.7 millimeter strainer and use forceps to manually massage the tissue. Place the contents back into the beaker and wash the tissue by covering it with 60 milliliters of deionized water per gram of tissue.

Stir at room temperature for 15 minutes. Repeat this wash process three times. After this, briefly dry the tissue on a delicate task wipe and weigh it. Transfer the tissue to a labeled 15 milliliter tube and freeze at negative 80 degrees Celsius overnight.